It is useful in a number of histochemical applications to be able to differentiate cell types based on their intracellular staining characteristics. Differential staining may reflect the presence or absence of particular organelles, or a difference in degree to which such organelles are present. For instance, the presence of nucleic acid or the amount thereof often serves as useful criteria for determining or distinguishing between various blood cell populations. Further, the ability to specifically stain various intracellular components may provide valuable pathological information concerning various disease states.
It is an object of the present invention to provide reagents and methods capable of exhibiting histochemical differentiation staining characteristics.
Several distinct classes of reagents have been employed to obtain useful intracellular staining. One such class relies solely upon the natural staining properties of dye materials as a means for providing specificity. A dye's natural staining properties refers to the specificity of molecule or cell stained by the dye. For example, the dye acridine orange has a natural specificity for staining DNA. Typically, these dyes are fluorophores which exhibit an emission spectra at a wavelength that is different than the wavelength used to excite the molecule. One example of such a dye is acridine orange and in U.S. Pat. No. 3,798,131, Rounds et al. describe a method for assaying polymeric DNA based on the nucleic acid staining properties of acridine orange.
Fluorophores, however, suffer from the disadvantage that any chemical modifications or manipulations to the fluorescent molecules intended to alter their inherent staining characteristics (i.e so that the dye can stain different classes or a wider range of molecules) also often result in substantial inactivation of the molecule's fluorescent properties. This inactivation apparently results from alteration of the critical three-dimensional structure of the fluorescent molecule which probably alters the molecule's energy transition levels. Consequently, fluorescent molecules often cannot be employed to their full advantage since the specificity of their natural staining characteristics cannot be directly altered without also severely reducing or destroying the molecule's fluorescent staining effectiveness. This is especially true with the so-called red dyes which typically fluoresce more weakly than the acridine orange or fluorescein dye families. Furthermore, chemical modification of the fluorophore intended to enhance direct binding to a target molecule is also problematic because of the same negative effect on the fluorophore's optical properties.
Despite characteristically weaker fluorescence, red dyes are still preferred in many histochemical applications because a less expensive helium-neon laser may be employed to excite the molecules to fluorescence as compared to the argon lasers necessary to excite fluorescein. Such commercial considerations become especially important with regard to instruments destined for use in the hospital and clinical laboratories where health care operating expenses are already at a premium. Unfortunately, the desirable red dyes not only suffer from weak fluorescence but, like all fluorescent molecules, any significant chemical manipulations designed to increase or improve their staining capability generally also results in serious deleterious effects on their fluorescent properties.
It is an object of the present invention to provide reagents and methods which allow for adjustment or manipulation of staining characteristics without incurring such deleterious associated loss of fluorescence.
One class of methods has attempted to capitalize on the immunological specifity associated with immunoglobulins as a means for altering the inherent staining characteristics of dyes. These methods rely on the immunoglobulin binding to a specific target molecule (thereby providing the desired specificity), wherein the fluorophore is bound to the immunoglobulin. Thus, direct binding of the fluorophore to the target molecule is avoided. One such method is described by Hirschfeld in U.S. Pat. No. 4,166,105 wherein a reagent is provided for the detection of a specific reactant. Such a reactant, generally an antigen, reacts immunologically only with the immunoglobulin which is the specific, complementary homolog for the antigen. By covalently linking a plurality of dye molecules to the immunoglobulin through a polymer such a polyethylene amine, the dye molecules stain materials based primarily on the specificity provided by the immunoglobulin. Thus, the dye molecules become associated only with those materials which express the antigenic determinants for which the antibody is specific.
Although such a reagent can often provide the desired staining specificity for staining, as well as localize a plurality of dye molecules at the desired site, such reagents are often difficult if not impossible to employ satisfactorily on an intracellular basis. This disadvantage is incurred due to the relatively large size of the resultant reagent; the antibody itself typically has a molecular weight of 150,000 daltons, further increased by the weight of the polyethylene amine (typically on the order of 20,000 daltons) plus the weight of a plurality of dye molecules which although comparatively far smaller on an individual basis (in the range of 50-2000 depending on the dye), becomes significant due to the 65 to 80 molecules expected per molecule of polyethylene amine. Such relatively large molecules encounter significant difficulty crossing cellular membranes. Accordingly, their effectiveness as an intracellular staining agent is significantly limited.
It is an object of the present invention to avoid such membrane penetrating limitations by providing reagents and methods which do not rely upon immunoglobulins for obtaining desired staining specificity.
It is a related object to provide reagents having the desired staining characteristics which are significantly smaller on a molecular weight basis than reagents employing immunoglobulin desired specificity.
It is another object to provide methods and reagents which can also localize a plurality of dye molecules at the site of interest whereby fluorescent signal amplification is effected.
Another approach employing immunoglobulin derived specificity has been described in U.S. Pat. No. 4,434,150 (Azad et al.). Therein, a plurality of dye molecules are linked to an immunoglobulin having the desired specificity by means of a polymer having specified charge and size characteristics. Because of the attachment of the relatively large immunoglobulin, however, such a reagent is similarly not preferred for many intracellular applications.
Still another approach attempting to improve intracellular staining is described by Scholefield in U.S. Pat. No. 4,094,745, wherein the microorganisms to be stained are treated chemically to modify the dye receptor sites in the microorganism. The thusly treated microorganisms are thereafter stained with a fluorochrome dye. Such methods, however, disadvantageously alter the natural properties of microorganisms and/or cells and fail to augment staining specificity associated with the dye. They also fail to provide signal enhancement by localization of a plurality of dye molecules at the desired site.
It is an object of the present invention to provide methods which employ reagents capable of carrying a plurality of dye molecules, such as the preferred red dye molecules, to intracellular sites in accordance with desired specificity characteristics without reliance upon immunoglobulin associated specificity or site altering solutions.